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. 2018 Aug 28;8(1):12946.
doi: 10.1038/s41598-018-31197-9.

A novel molecular rotor facilitates detection of p53-DNA interactions using the Fluorescent Intercalator Displacement Assay

Walter L Goh  1 , Min Yen Lee  2 , Ting Xiang Lim  1 , Joy S Chua  1 , Sydney Brenner  2 , Farid J Ghadessy  3 , Yin Nah Teo  4
Affiliations

A novel molecular rotor facilitates detection of p53-DNA interactions using the Fluorescent Intercalator Displacement Assay

Walter L Goh et al. Sci Rep. .

Abstract

We have investigated the use of fluorescent molecular rotors as probes for detection of p53 binding to DNA. These are a class of fluorophores that undergo twisted intramolecular charge transfer (TICT). They are non-fluorescent in a freely rotating conformation and experience a fluorescence increase when restricted in the planar conformation. We hypothesized that intercalation of a molecular rotor between DNA base pairs would result in a fluorescence turn-on signal. Upon displacement by a DNA binding protein, measurable loss of signal would facilitate use of the molecular rotor in the fluorescent intercalator displacement (FID) assay. A panel of probes was interrogated using the well-established p53 model system across various DNA response elements. A novel, readily synthesizable molecular rotor incorporating an acridine orange DNA intercalating group (AO-R) outperformed other conventional dyes in the FID assay. It enabled relative measurement of p53 sequence-specific DNA interactions and study of the dominant-negative effects of cancer-associated p53 mutants. In a further application, AO-R also proved useful for staining apoptotic cells in live zebrafish embryos.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Chemical structures of the molecular rotors used. Shown below are synthetic routes for compounds II, III and IV.
Figure 2
Figure 2
Fluorescence response of molecular rotors in the presence of DNA. (A–G) Fluorescence signal of rotor molecules at a fixed concentration (100 nM) titrated against increasing concentrations of double-stranded DNA fragments. Black-bar insets show fluorescence signal at maximum measurement configuration and highest DNA concentration. Hashed-lines depict levels of dye-only background fluorescence. (H) Levels of turn-on fluorescence from increasing concentrations of each compound in the presence of a fixed concentration of DNA (200 nM). All data shows mean ± S.D. of 3 individual experiments. For Py-R with dual excitation wavelengths, results for 376 nM wavelength shown. Similar results were observed upon excitation at 342 nM (Supplementary Fig. 2).
Figure 3
Figure 3
Signal intensity heat map of DNA-induced and sequence-dependent fluorescence intensity across rotor compounds. Color representative heat map showing rotor-specific fluorescence response to a panel of DNA oligonucleotide sequences containing p53 response elements and control sequence (scram). Color spectrum depicts absolute amount of fluorescence gained for each dye in the presence of DNA (color scale bar: violet = self-referenced dye-only signal, red = highest observed Δa.u. across all dyes, set at 10 000 a.u.). Each row depicts fluorescence signals from individual compounds with lower limit (violet) benchmarked to respective “dye only” background signals.
Figure 4
Figure 4
Fluorescent intercalator displacement assay with rotor compounds. Percentage change in fluorescence intensity when purified wildtype-p53 protein is added to mixtures containing rotor pre-complexed with different DNA fragments. Black-hash line depicts 100% reference value (rotor and DNA only), below which shows a displacement event. Grey-hash line shows percentage-change in signal for scrambled-DNA sequence. Data shows mean ± S.D. for 2 biological replicates.
Figure 5
Figure 5
Acridine orange rotor (AO-R) as a fluorescence probe for sequence-specific p53-DNA binding. (a) FID assay using purified wildtype p53 protein at indicated concentrations on response elements RGC, p21, and scrambled control DNA (black bars) pre-complexed with AO-R. Data normalized to scram control showing FID effects through sequence-specific DNA binding. (b) FID assay using either wildtype p53, p53 G245S mutant or p53 R2733H mutant (10 µM) on DNA response elements pre-mixed with AO-R. (c) FID assay using AO-R to examine dominant effects of mutant p53 proteins, which were first prepared to contain different proportions of wildtype and mutant p53 molecules (10 µM final). Left: FID response of AO-R pre-complexed RGC-RE exposed to p53 mixtures containing wildtype and G245S mutant proteins. Right: FID response of AO-R pre-complexed p21-RE exposed to p53 mixtures containing wildtype and R273H mutant proteins. Black bar on right shows RE-AO-R complex reference signal at 100% (black hash line). All data shows mean ± S.D. of 3 individual FID experiments.
Figure 6
Figure 6
In-vivo staining of live zebrafish embryos using compound AO-R. (a) Live imaging of either wildtype (top panels), or Mdm−/− (bottom panels) 1 day post fertilization zebrafish embryos stained using either AO-C (left panels) or AO-R (right panels) dyes. Scale bars measure 1 mm. (b) Fluorescence intensity of individually measured whole, wildtype or Mdm2 knock-out, zebrafish embryos stained with either AO-C or AO-R dyes. (N = 22; *P < 0.0001, unpaired, two-tailed student t test).
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